Antimicrobial peptides play a major role in humoral innate immunity against microorganisms. Multiple in vivo and in vitro studies highlight the important function of human group IIA secreted phospholipase A2 (hGIIA) in protection against Gram-positive bacterial infection including Group B Streptococcus (GBS), a leading cause of neonatal sepsis and meningitis. hGIIA kills bacteria by catalyzing the hydrolysis of the membrane glycerophospholipids. The Gram-positive cell envelope, consisting of multiple peptidoglycan layers decorated with a variety of glycopolymers, represents a substantial barrier to hGIIA and it is poorly understood how hGIIA gains access to the bacterial plasma membrane to produce lethal damage. We have recently identified novel bacterial resistance mechanisms against hGIIA in the bacterium closely-related to GBS, Group A Streptococcus (GAS). Our work revised the current models of GAS cell wall architecture and highlighted the importance of these structural determinants for resistance to hGIIA. Interestingly, GBS is killed by hGIIA at concentrations that are approximately 500-fold lower, than the GAS strains. The experiments described in this proposal are designed to understand the underlying mechanisms for hGIIA potency against GBS. To accomplish our goal, we will use two approaches. In the first approach we will conduct screens of a recently constructed, highly saturated GBS transposon mutant library using lethal and sub-lethal concentrations of hGIIA. After construction of deletion mutants in the identified genes we will perform antimicrobial and mechanistic assays and transmission electron microscopy analysis of the freeze-substituted cells to confirm the hGIIA susceptibility phenotype of the mutants and understand the mechanisms of resistance/susceptibility. In the second approach, we will investigate the role of the major peptidoglycan-attached glycopolymer of GBS, the Group B Carbohydrate (GBC), in hGIIA susceptibility by swapping GBC with the GAS cell wall glycopolymer in GBS. Successful outcomes will guide future efforts for new drug development against GBS.